Cancer immunotherapy, particularly genetically engineered adoptive T cell transfer, has shown great potential for the treatment of cancer patients. The use of T cells engineered to express a specific T cell receptor (TCRs) or chimeric antigen receptor (CARs) to treat cancer has generated durable cures for many cancer patients and has resulted in the first FDA approved CAR-T therapy to treat childhood acute lymphoblastic leukemia in 2017. Most gene therapies rely on viral methods to genetically modify human primary cells. However, viral delivery method is expensive, poorly reproducible and associated with several safety concerns including insertion in or near genes that may cause malignancy and generation of replication competent virus. Thus, non-viral DNA delivery methods, such as Sleeping Beauty and piggyBac, have been employed to generate CAR T cells. Although these non-viral delivery methods have the advantage of lower cost, immunogenicity, and regulatory considerations, they have been limited by their low transposition efficiency in primary human hematopoietic cells. In this application, we propose to rationally optimize a recently discovered transposon, TcBuster to deliver CARs to T cells. To this end, we further enhance our already very active hyperactive mutants of the TcBuster transposase and optimize the delivery of the transposon into cells. Following optimization of the TcBuster transposon system, we will combine these improvements with our proprietary methods to transfect T-cells efficiently and safely, and test the immunotherapeutic effectiveness of TcBuster delivered CAR into T cells. The successful completion of this project will result in the comprehensive methods to produce CAR T cells delivered by TcBuster which we will license to pharmaceutical companies to produce highly efficient CAR-T immunotherapy. More broadly, these methods could be expanded beyond immunotherapeutic cancer applications to various infectious diseases in which gene delivery by TcBuster in T cells could be advantageous.